The present invention relates to a dry chromatography method for the purification of from 1 mg to a few hundred mg quantities of compounds, to the type of columns and to the nature of the sorbents to be used for such separations. The method is suitable for purification of several compounds simultaneously, e.g. the purification of chemical libraries. As described in the present invention the method is well suited for automation since addition of the sample can be done in a simple and reproducible manner and since the eluent is simply added to the top of the column and in the exact amount needed for the separation.
Combinatorial chemistry and high speed synthesis has become very important tools in medicinal chemistry. Large xe2x80x9clibrariesxe2x80x9d of compounds are synthesised and subjected to High Throughput Screening (HTS) trying to identify novel structures with interesting biological activity. A xe2x80x9cchemical libraryxe2x80x9d may contain several thousands of compounds.
The chemical libraries may be prepared by conventional solution chemistry but solid phase methods are also being used. Many different types of automated synthesisers have been developed and automation is indeed a very important factor when trying to handle large numbers of compounds simultaneously. The compounds are commonly prepared in amounts of 1-100 mg, most often about 5-20 mg.
The quality of the library is highly dependent on the purity of the individual compounds present in the library. When a set of chemical reactions are applied to different substrates the yields of reaction may differ significantly, the end result being a chemical library where the purity of the compounds may vary considerably. Obviously, a simple method for the purification of several compounds at the same time would be very attractive. The method should preferably be suited for automation.
HPLC systems equipped with automated injectors can handle the separation of several samples. However the samples are separated sequentially on the same column and the column has to be regenerated after each run. Large amounts of solvents are needed and contamination of the column may often be a serious problem preventing long time use. Also, an extreme number of different fractions need to be collected and handled.
Dry column chromatography is a relatively old chromatographic method (Reviews: B. Loev and M. M. Goodman. in xe2x80x9cProgress in Separation and Purificationxe2x80x9d 1970; vol. 3: 73-95. F. M. Rabel in M. Zeif and R. Speights (Eds.), xe2x80x9cUltrapuificationxe2x80x9d, Dekker, New York 1972, p 157). The mixture to be purified is added to the top of a dry column of e.g. silica and the column is developed with a suitable solvent. When the solvent reaches the end of the column the separated compounds are located (e.g. by colour), the column cut into pieces and the desired compounds isolated by extraction. Typically column sizes of diam. 1-3 inches and length 10-20 inches have been used for purification of compound mixtures of several hundred mg to several grams. The particle size used has commonly been  greater than 63xcexc.
Dry column chromatography has not been utilised for the simultaneous purification of large numbers of compounds, e.g. the purification of chemical libraries, and has never been subjected to automation in this connection.
Dry column chromatography has not been recognised as a suitable method for rapid purification of mg quantities of compounds on short columns.
As mentioned above dry column chromatography is an old chromatographic method and today it is practically never used. Obviously the advantages that are obtained when it is performed as described in the present invention have not been realised.
The object of the present invention is to eliminate the shortcomings and solve the problems stated above in connection with the known techniques for simultaneous purification of several compounds in a sample. This object is achieved by a chromatografic column of the type described by way of introduction having the features specified in the characterising part of claim 1. These and other features and advantages will appear from the following description and the subclaims. The present invention also relates to a method for simultaneous purification of several compounds in a chromatography column according to the present invention and also to use of such a column.
The present invention relates to a dry chromatography column to be used for rapid purification of from about 1 mg to a few hundred mg quantities of compounds. Several separations can be run simultaneously and the columns are very well suited for use in automated procedures.
According to the. present invention it has been found that short dry chromatography columns can be prepared by packing columns of suitable plastic materials with sorbents of small particle size, and that these columns can be used for rapid purification of small amounts of material. The top of the column contains an empty volume. Methods are provided whereby addition of the sample can be done in a simple and reproducible manner. The eluent is thereafter added to the top of the column and in the amount needed for the separation. The simplicity and the reproducibility of the chromatographic method described in the present invention make it well suited for automation. When the separations have been completed the desired compounds can be located on the columns, that particular part of the column cut out, and the compounds isolated by extraction.
Compared to conventional HPLC methods dry column chromatography utilises only small amounts of solvents and does not require handling of very large numbers of different fractions.
The columns of the present invention can be made of different plastic materials. These materials should be transparent or semitransparent to UV and visible light, to allow for location of the separated compounds on the column. The plastic should be compatible with the solvents to be used for the separations. Since the compounds are isolated by cutting out the desired part of the column it is important that the plastic is easy to cut with minimal deformation of the sorbent. Suitable thicknesses are between 0.5 to 3 mm, preferably 1 to 2 mm. Since the columns of the present invention are short they are still rigid enough to be easy to handle. Cutting can be done with a sharp object such as a knife, razorblade and the like. The columns are preferably cylindrical but other forms may be used.
Suitable plastic materials are those which can be used together with different organic solvents e.g. polyethylene, polypropylene and Teflon. Teflon has superior chemical resistance and is soft to cut. Polyethylene is softer than polypropylene. If very soft plastic materials are used, such columns may be put inside an outer shell. Combinations of plastic materials may also be used.
For the separations to proceed with acceptable speed the length of the column is generally less than 20 cm, preferably less than 15 cm. Columns of small diameter are easier to cut without deformation than wider columns. The diameter of the column may generally vary from about 0.3 to about 3 cm, preferably less than 1.5 cm. However, smaller columns may be used if almost analytical separations are desired and larger columns may be of use for separations of large amounts of material. For large amounts of material it may be more advantageous to use several columns in parallel. The problem that a compound moves slightly faster near the column wall is of greater significance for wider columns (c.f. B. Loev and M. M. Goodman. in xe2x80x9cProgress in Separation and Purificationxe2x80x9d 1970; vol. 3: 73-95).
The column should have an empty volume above the sorbent preferably large enough to be able to hold the volume of solvent needed to develop the column. For a silica column this volume is generally 0.5 to 0.8 times the volume holding the sorbent. The volume may be indicated by a mark, a helpful feature when adding the developing solvent.
It is very important that the sample to be purified can be added to the column in a simple and reproducible manner, e.g. from a syringe or a pipette, so that it forms a narrow and uniform band at the top of the column. This is particularly difficult to accomplish for small columns. However, according to the present invention this problem has been solved by different methods as described below.
A portion of inert material without retaining capacity can be put on top of the sorbent. The sample, dissolved in a small amount of solvent is added and absorbs into the inert material. Subsequent addition of developing solvent transports the sample to the top of the sorbent where it forms a uniform band, whereafter development of the column proceeds in the normal manner. Inert materials, which can be used for the purpose above, are materials, which do not retain the sample and may e.g. be a plastic material such as polyamide. The inert material functions as a concentration zone. A thick filter made of similar materials may be used for the same purpose. The use of a small amount of relatively unpolar developing solvent in the beginning may be advantageous in order to minimise the band.
A different way to accomplish a uniform band at the top of the column involves the use of a plug, having one or several small holes, which covers the top of the column. The sample, dissolved in a small amount of solvent, is added to the column and is relatively slowly absorbed through the small holes onto the column in such a way that it forms a uniform band. The size of the holes, the number of holes and the exact shape of the plug may vary and is e.g. dependant on the diameter of the column, it being important that absorption onto the column proceeds in a uniform manner. The plug is preferably made of an inert plastic material and the thickness is typically 0.5-6 mm. The plug may be an integral part of the column.
Another opportunity is to, on top of the column, use a porous filter, which has the capability to absorb and distribute the sample solution within itself, before the solution enters onto the top of the sorbent. Such filters may e.g. be made of different plastic materials. Filters of polyethylene may be very useful.
Still another opportunity is to absorb the sample into a porous plug, which can subsequently be put on top of the column. The plug should cover the surface of the column and should be made from a material, which does not retain the compounds when the eluting solvent is added, the compounds being transferred directly onto the top of the column.
A preferred way to accomplish a uniform addition of the sample is to first xe2x80x9cwetxe2x80x9d the column with a small amount of solvent. Subsequent addition of the sample solution allows It to be absorbed onto the column more slowly and this in turn results in a uniform band. This type of sample addition has not been described previously in combination with dry column chromatography.
Various combinations of the methods described above and which give a uniform band on top of the column may also be used.
It is important that the column can be cut into pieces in a reproducible manner and that the sorbent is retained in the piece that is cut out. According to the present invention it has been found that this is best accomplished when the sorbent is of small particle size and that particles of irregular form may stick together better than spherical particles. The problem is especially relevant when volatile solvents are being used to develop the column, since when the solvents evaporate the piece of column which is cut out dries. For columns with large diameters the problem may be of greater importance than for columns with small diameters.
As is evident from that stated above, the particle size of a sorbent in the column according to the present invention is of great importance, and several factors have to be considered and balanced with a view to obtaining good separation characteristics of the column. It is well known that sorbents having small particle sizes are difficult to dry pack, as aggregates are formed leaving too long an eluation time. In contrast, sorbents having large particle sizes are easier to dry pack, resulting in columns with a shorter eluation time. However, in the latter case the separation characteristics are impaired, and when the column is cut into pieces, the sorbent is likely to fall out of the column.
The inventor has surprisingly found that columns having dimensions here defined can be dry packed in a satisfactory way with sorbents having a small particle size. In particular, Grace silica with a particle size of 6-35 xcexcm has appeared to give very favourable flow characteristics, probably due to a small amount of very small particles.
When silica is used as sorbent it is preferred that the particle size is  less than 63xcexc and  less than 40xcexc is even more preferable. Silica""s with particle sizes ranging between 5-40xcexc or 6-35xcexc are suitable. It is important to choose a silica which allows the columns to be developed as rapidly as possible. This also means that longer columns can be produced. According to the present invention it has surprisingly been found that a silica of particle size 6-35xcexc from Grace is much superior to a Matrex silica of particle size 6-35xcexc from Amicon which in turn is superior to a silica of particle size 5-40xcexc from Merck. When examined in the microscope the Grace silica seem to contain a minimum of very fine particles and this might explain its superior properties when used for dry column chromatography purposes. A column packed with silica from Grace is developed about 50% faster than a column with Matrex silica from Amicon. Yet another advantage with the Grace silica is that it is easier to pack than the other ones. Still another advantage of this silica is that it is available at a low price, which is important since the columns are only utilised once.
When aluminium oxide is used as sorbent, similar particle sizes  less than 63xcexc and  less than 40xcexc are also suitable.
Sorbents having small particle size generally give sharper separations. For this reason a silica with particle size  less than 25xcexc e.g. 15-25xcexc, or even better 5-20xcexc, may be suitable.
Sorbents having small particle size and having spherical particles give good separations.
Silica and aluminium oxide are preferred sorbents, but in principle all sorbents used for chromatography can be utilised in the present invention. The silica or aluminium oxide is preferably deactivated (c.f. B. Loev and M. M. Goodman. in xe2x80x9cProgress in Separation and Purificationxe2x80x9d 1970; vol. 3: 73-95). When the sorbent is deactivated in the open air the amount of water which is absorbed may vary depending on e.g. humidity.
The sorbents may have been pre-treated in various ways to make them suitable for different type of separations. E.g. they may have been conditioned in the presence of suitable chemicals. Silica to be used for the separation of amines may thus be conditioned in vapour containing ammonia or other volatile amines. Another way to condition the column is to put the ready column in such vapours. In this case the conditioning process may be accelerated by drawing, e.g. using vacuum, amine vapours through the column.
It is preferred that the sorbents (c.f. above) and the different parts of the columns (e.g. filters) are available at relatively low prices since they are only utilised once.
Various binders can be used in order to xe2x80x9cgluexe2x80x9d the sorbent together and thus improve the properties of the column regarding performance when being cut. Calcium sulphate is a binder often used in connection with TLC, and is useful for the purpose above. The use of a binder and the amount of binder to be used may vary according to the specific application, but generally binders may be of greater importance for columns of wider diameter.
It is important that when a mixture has been separated on the column that the individual components can be visualised. This may be accomplished by well known techniques used e.g. in TLC. Commonly a fluorescent material is incorporated into the sorbent, giving dark bands where UV absorbing components are located. Some compounds may be fluorescent by themselves when irradiated with light of appropriate wavelength, and of course coloured compounds are easily seen. For compounds, which do not absorb UV-light, the position of the compound may be estimated by comparison with TLC Rf-values. Another opportunity when the compounds cannot be visualised is to cut the column in pieces and analyse each piece individually.
The columns may be packed by known dry packing techniques. Uniform and dense packing is important for satisfactory chromatographic performance. The columns can e.g. be packed by simultaneous addition of sorbent and tapping the bottom of the column firmly against the surface. Tapping is continued until the sorbent is densely packed. According to the present invention it has been found that a Grace silica of particle size 6-35xcexc is very easy to dry pack. Vacuum may be applied during packing. Pressure may also be used. The sorbent is confined on the column by filters on the top and at the bottom.
It is generally believed in the art that sorbents containing particles  less than 20xcexc cannot be dry packed. However according to the present invention it has been found that this type of packing works well for dry chromatography, especially for the Grace silica described above.
The columns may have a scale on the outside. This makes it possible to determine Rf-values, which may be used as part of compound identity, and may be of help when examining the column in order to decide which pieces to cut out.
The solvents used to develop the column may be all those known in the art. It has been stated (B. Loev and M. M. Goodman. in xe2x80x9cProgress in Separation and Purificationxe2x80x9d 1970; vol. 3: 73-95) that the use of solvent mixtures may be disadvantageous. According to the present invention it has been found that solvent mixtures work well.
It is of some importance to analyse the column and to cut out the desired fractions directly after it has been developed. Otherwise the compounds will diffuse and this will result in band broadening.
In a modification of the present invention the sample to be separated is allowed to be absorbed into the filter and sorbent at the bottom of the column. Development of the column is subsequently achieved by putting the column in the desired development solvent and allowing this to ascend through the column by capillary action. For these columns it is of course not necessary that they have an empty volume on top of the column.
It is important to be able to cut the columns into small pieces with a minimum of deformation. Cutting devices that are able to provide support around the column are suitable for this purpose and these are also within the scope of the present invention. A preferred cutting device is characterised in that it supports the column on both sides of the cutting zone.
The type of chromatography described in the present invention is highly suited for automation. Various robots and machines can be used to add samples and developing solvents to large numbers of columns. The use of automation together with the present type of chromatography is also within the scope of the present invention.
The handling of several columns simultaneously is of great interest in connection with purification of chemical libraries and this particular utilisation lies within the scope of the present invention. For combinatorial libraries it may not be necessary to obtain extremely pure compounds but purities of 80-90% may be sufficient. The present invention is very useful for this purpose.